Co-encapsulation of live cells with oxygen-generating particles

ABSTRACT

Microcapsules are described that comprise (a) a liquid aqueous or hydrogel core; (b) a semipermeable membrane surrounding said core; (c) live animal cells (e.g., pancreatic cells) in the core; and (d) oxygen-generating particles in said core, said oxygen-generating particles included in said microcapsules in an amount sufficient to lengthen the duration of viability of said animal cells in said microcapsules. Compositions comprising such microcapsules and uses thereof, such as in treating diabetes, are also described.

RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/237,214, filed May 13, 2014, now allowed, whichis a 35 U.S.C. §371 national phase entry of PCT ApplicationPCT/US2012/050060, filed Aug. 9, 2012, and published in English on Feb.14, 2013, as International Publication No. WO 2013/023013, and whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/521,420, filed Aug. 9, 2011, and U.S. Provisional Patent ApplicationSer. No. 61/601,780, filed Feb. 22, 2012, the disclosure of each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Glycemic control in diabetes has been shown to delay the onset of, andslow the progression of, associated pathological complications. However,achieving adequate glycemic control using insulin therapy can bedifficult. One alternative to insulin therapy is the transplantation offunctioning pancreatic islet cells to diabetic subjects, to providebiological insulin replacement. However, transplanted or grafted isletcells encounter immunological rejection, which can limit the clinicalusefulness of his method. Microencapsulation of islet cells has beenproposed to reduce or avoid immunological rejection of transplantedislet cells. See, e.g., U.S. Pat. No. 6,783,964 to Opara. There remainsa need, however, for new ways to facilitate the effective implantationof live encapsulated pancreatic islet cells for the treatment ofdiabetes.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a microcapsule comprising:(a) a liquid aqueous or hydrogel core; (b) a semipermeable membranesurrounding that core; (c) live animal cells in the core; and (d)oxygen-generating particles in said core, the oxygen-generatingparticles included in said microcapsules in an amount sufficient tolengthen the duration of viability (e.g., lengthen the life-span) of theanimal cells in the microcapsules.

In some embodiments, the oxygen-generating particles comprise abiodegradable polymer (e.g., from 0.1 to 15 or 30 percent by weight) incombination with an inorganic peroxide (e.g., from 40 to 99 percent byweight).

In some embodiments, the inorganic peroxide is selected from the groupconsisting of calcium peroxide, magnesium peroxide, sodium peroxide,sodium percarbonate, benzylperoxide, and combinations thereof.

In some embodiments, the oxygen-generating particles and/or themicrocapsules further comprise an antioxidant, a radical trap, and/or aperoxide decomposition catalyst (e.g., (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl, catalase, ascorbic acid)(e.g., from zero to 30 percent by weight).

In some embodiments, the microcapsules further comprising an auxiliarylayer surrounding the semipermeable layer.

In some embodiments, the auxiliary layer comprises a biodegradablepolymer, said biodegradable polymer optionally containing an activecompound (e.g. angiogenic proteins, antibodies, hormones, polypeptides,amino acids, neurotransmitters, neurotoxins, viruses, cytokines, andnucleic acids).

In some embodiments, the said auxiliary layer contains a pro-angiogenicagent in an amount effective to enhance the growth of blood vesselsadjacent said microcapsule (e.g., after in vivo implantation thereof ina mammalian subject, such as for carrying out a method as describedherein).

In some embodiments, the animal cells are mammalian cells.

In some embodiments, the mammalian cells are pancreatic islet cells,ovarian cells (e.g., granulosa and/or theca cells) hepatocytes,myocytes, alveoli, neuronal cells, and/or adrenal cells.

A further aspect of the invention is a composition comprising orconsisting essentially of microcapsules as described herein in apharmaceutically acceptable carrier.

A further aspect of the invention is a method of treating diabetes in asubject in need thereof, comprising implanting microcapsules asdescribed herein in said subject in a treatment-effective amount.

A further aspect of the invention is microcapsules as described hereinfor use in treating diabetes in a subject in need thereof.

Implantation of oxygen-generating particles is described, for example,in U.S. Patent Application No. US 2010/0112087 to Harrison et al. (May6, 2010), for the purpose of treating hypoxic tissue. However, theco-encapsulation of oxygen generating particles with live cells isneither suggested nor described.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below. The disclosures of allUnited States patent references cited herein are to be incorporated byreference herein in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of CaO₂ and MgO₂ release over six days, dissolvedoxygen readings are an average of 5 sample groups with 1500microcapsules each; measurements were taken in a hypoxic chamber.

FIG. 2: Effect of antioxidants on oxygen release from alginatemicrocapsules with 2.5% MPO. In the presence of 100 U/ml catalase and100 U/ml Catalase+1.5 mM Trolox the oxygen release is significantlyshortened compared to the release of MPO alone. However this effect isnot observed with 1.5 mM Trolox alone.

FIG. 3: Quantative analysis of confocal images. Each group is theaverage of 3 cells selected at random, error bars represent standarddeviation.

FIG. 4: Viability of islets co-cultured with MPO and antioxidants.Isolated human islets were co-cultured with 10 mM MPO for 2 days innormal culture conditions with the following antioxidants: 100 Units/mLCatalase, 1.5 mM Trolox and 30 μM MnTE-2-Pyp. After the two day cultureperiod islet viability was determined with an MTS assay. Error barsrepresent standard deviation, n=5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Subjects” as used herein are, in general, mammalian subjects. Whilehuman subjects are preferred, the subjects may in some embodiments beother animals, such as dogs and cats for veterinary purposes. Subjectsare generally female. While the subjects may be of any suitable age, thesubjects are typically adults and in some embodiments are menopausalfemale subjects.

“Treat” as used herein refers to any type of treatment that imparts abenefit to a subject, including but not limited to delaying the onset orreducing the severity of at least one symptom in the subject

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Biologically active compound” as used herein may be any suitablecompound, including but not limited to TGF-beta, basic fibroblast growthfactor (FGF2), epithelial growth factor (EGF), insulin-like growthfactor-1 (IGF-1), transforming growth factors alpha and beta (TGF-1alpha and beta), nerve growth factor (NGF), platelet-derived growthfactor (PDGF), vascular endothelial growth factor/vascular permeabilityfactor (VEGF/VPF), anti-virals, anti-bacterials, anti-inflammatory,immuno-suppressants, analgesics, vascularizing agents or pro-angiogenicagents, and cell adhesion molecules, and combinations thereof. See,e.g., US Patent Application No. 20110052715 (Mar. 3, 2011).

1. Cells.

Cells used to carry out the present invention are, in general, livemammalian cells collected from a suitable donor. Donors are, in general,mammalian (e.g., human, dog, cat, rabbit, rat, mouse, monkey,chimpanzee, horse, pig, goat, sheep). The donor may be of the samespecies as the subject being treated, or of a different species. In someembodiments the donor may be the same subject undergoing treatment,where suitable cells were harvested from the subject and stored forsubsequent use.

Cells are isolated from donors and cultured for microcapsule productionas desired in accordance with techniques known in the art. See, e.g.,Sanjay K. Agarwal et al., Leptin Antagonizes the Insulin-Like GrowthFactor-I Augmentation of Steroidogenesis in Granulosa and Theca Cells ofthe Human Ovary, J. Clin Endocrinol Metab 84: 1072-1076 (1999); Jon C.Havelock et al., Ovarian granulosa cell lines, Molecular and CellularEndocrinology 228, 67-78 (2004); Jessica K. Wickenheisser et al., Humanovarian theca cells in culture, Trends in Endocrinology & Metabolism 17,65-71 (2006). In general, fresh tissue is divided by mincing, teasing,comminution and/or collagenase digestion. The desired cells are thenisolated from contaminating cells and materials by washing, filtering,centrifuging or picking procedures, and optionally cultured and/orcryopreserved as desired prior to encapsulation.

2. Oxygen-Generating Particles.

Any suitable oxygen-generating particle can be used to carry out thepresent invention, including but not limited to encapsulated hydrogenperoxide, inorganic peroxides, or peroxide adducts such as described inUS Patent Application Publication Nos. 2009/0169630 to Ward et al. and2010/0112087 to Harrison et al. (the disclosures of which areincorporated by reference herein in their entirety). Theoxygen-generating particles preferably comprise an organic or inorganicperoxide such as urea peroxide, calcium peroxide, magnesium peroxide,and/or sodium percarbonate. The oxygen-generating active agent isincluded in the composition in any suitable amount (e.g., from 0.1 or 1to 10, 20, or 30 percent by weight, or more). In some embodimentscalcium peroxide is preferred as it releases oxygen at a desireable ratein situ. The oxygen-generating active agent can be included in thepolymer in solid form, such as in the form of a plurality of solidparticles thereof.

In some embodiments a radical trap or peroxide or radical decompositioncatalyst is also included in the oxygen-generating particle and/or themicrocapsule composition (e.g., in an amount of from 0.1 or 1 to 10, 20or 30 percent by weight, or more). Suitable examples of radical traps ordecomposition catalysts include, but are not limited to, iron(including, but not limited to, iron particles or nanoparticles, enzymessuch as catalase, peroxidase, or dehydrogenase (see, e.g., U.S. Pat. No.7,189,329), compounds such as cyclic salen-metal compounds that havesuperoxide and/or catalase and/or peroxidase activity (see, e.g., U.S.Pat. No. 7,122,537), etc.). The radical trap or decomposing catalyst maybe included in solid form (e.g., solid particulate form) and can becoated on or incorporated in the polymer, or both coated on andincorporated in the polymer).

3. Microcapsule Production.

Encapsulation of live cells in combination with oxygen-generatingparticles can be carried out in accordance with known techniques orvariations thereof that will be apparent to those skilled in the art.See, e.g., U.S. Pat. Nos. 6,783,964 and 6,365,385 to Opara, thedisclosures of which are incorporated by reference herein in theirentirety.

Microcapsules useful in the present invention optionally, but in someembodiments preferably, have at least one semipermeable membranesurrounding a cell-containing interior (preferably a hydrogel interior).The semipermeable membrane permits the diffusion of nutrients,biologically active molecules and other selected products through thesurface membrane and into the microcapsule core. The surface membranecontains pores of a size that determines the molecular weight cut-off ofthe membrane. The membrane pore size is chosen to allow the passage ofactive agents secreted by the cells (e.g., insulin from pancreaticcells; estrogen, and in some embodiments progesterone, from ovariancells; etc.) from the within the capsule to the external environment,but to exclude the entry of host immune response factors (where theencapsulated cells are not autologous). Such a semipermeable membrane istypically formed from a polycation such as a polyamine (e.g., polylysineand/or polyornithine), as discussed further below.

In one non-limiting example embodiment of an encapsulation technique,U.S. Pat. No. 4,391,909 to Lim et al describes a method in which cellsare suspended in sodium alginate in saline, and droplets containingcells are produced. Droplets of cell-containing alginate flow intocalcium chloride in saline. The negatively charged alginate dropletsbind calcium and form a calcium alginate gel. The microcapsules arewashed in saline and incubated with poly-L-lysine or poly-L-ornithine(or combinations thereof); the positively charged poly-l-lysine and/orpoly-L-ornithine displaces calcium ions and binds (ionic) negativelycharged alginate, producing an outer poly-electrolyte semipermeablemembrane. An exterior coating of sodium alginate may be added by washingthe microcapsules with a solution of sodium alginate, which ionicallybonds to the poly-L-lysine and/or poly-L-ornithine layer (this serves toreduce any inflammatory response that may be provoked in the subject bycontact of the polycationic membrane to tissue). This technique produceswhat has been termed a “single-wall” microcapsule. A “double-wall”microcapsule can be produced by following the same procedure as forsingle-wall microcapsules, but prior to any incubation with sodiumcitrate, the microcapsules are again incubated with poly-l-lysine andsodium alginate.

In additional non-limiting examples of encapsulation methods, Chang etal., U.S. Pat. No. 5,084,350 discloses microcapsules enclosed in alarger matrix, where the microcapsules are liquefied once themicrocapsules are within the larger matrix. Tsang et al., U.S. Pat. No.4,663,286 discloses encapsulation using an alginate polymer, where thegel layer is cross-linked with a polycationic polymer such aspolylysine, and a second layer formed using a second polycationicpolymer (such as polyornithine); the second layer can then be coated byalginate. U.S. Pat. No. 5,762,959 to Soon-Shiong et al. discloses amicrocapsule having a solid (non-chelated) alginate gel core of adefined ratio of calcium/barium alginates, with polymer material in thecore. U.S. Pat. Nos. 5,801,033 and 5,573,934 to Hubbell et al. describealginate/polylysine microspheres having a final polymeric coating (e.g.,polyethylene glycol (PEG)); Sawhney et al., Biomaterials 13:863 (1991)describe alginate/polylysine microcapsules incorporating a graftcopolymer of poly-l-lysine and polyethylene oxide on the microcapsulesurface, to improve biocompatibility; U.S. Pat. No. 5,380,536 describesmicrocapsules with an outermost layer of water soluble non-ionicpolymers such as polyethylene(oxide). U.S. Pat. No. 5,227,298 to Weberet al. describes a method for providing a second alginate gel coating tocells already coated with polylysine alginate; both alginate coatingsare stabilized with polylysine. U.S. Pat. No. 5,578,314 to Weber et al.provides a method for microencapsulation using multiple coatings ofpurified alginate. U.S. Pat. No. 5,693,514 to Dorian et al. reports theuse of a non-fibrogenic alginate, where the outer surface of thealginate coating is reacted with alkaline earth metal cations comprisingcalcium ions and/or magnesium ions, to form an alkaline earth metalalginate coating. The outer surface of the alginate coating is notreacted with polylysine. U.S. Pat. No. 5,846,530 to Soon-Shiongdescribes microcapsules containing cells that have been individuallycoated with polymerizable alginate, or polymerizable polycations such aspolylysine, prior to encapsulation.

When desired, the alginate-polylysine microcapsules can be incubated insodium citrate to solubilize any calcium alginate that has not reactedwith poly-l-lysine, i.e., to solubilize the internal core of sodiumalginate containing the cells, thus producing a microcapsule with aliquefied cell-containing core portion. See Lim and Sun, Science 210:908(1980). Such microcapsules are referred to herein as having “chelated”,“hollow” or “liquid” cores.

When desired, the microcapsules may be treated or incubated with aphysiologically acceptable salt such as sodium sulfate or like agents,in order to increase the durability of the microcapsule, while retainingor not unduly damaging the physiological responsiveness of the cellscontained in the microcapsules. See, e.g., U.S. Pat. No. 6,783,964 toOpara.

One currently preferred method for the production of microcapsules isdescribed in O. Khanna et al., Synthesis of multilayered alginatemicrocapsules for the sustained release of fibroblast growth factor-1 J.Biomed. Mater. Res. Part A:95A: 632-640 (2010).

Microcapsules may be of any suitable size, such as from 10, 20 or 30microns in diameter, up to 1000, 2000, or 5000 microns in diameter.Microcapsules may contain any suitable amount of cell. For example, insome embodiments, the cells are included in the microcapsules in anamount of from 1,000 or 2,000 cells per microcapsule up to 1×10⁶, 1×10⁸,or 1×10⁹ cells per microcapsule; and the theca cells are included in themicrocapsules an amount of from 1,000 or 2,000 cells per microcapsule upto 1×10⁶, 1×10⁸, or 1×10⁹ cells per microcapsule.

In some embodiments an antioxidant is also included in the microcapsuleand/or the oxygen-generating particle composition (e.g., in an amount offrom 0.1 or 1 to 10, 20 or 30 percent by weight, or more). Suitableexamples of antioxidants include, but are not limited to, ascorbic acidor vitamin C, tocopherols and tocotrienols such as vitamin E and analogsthereof such as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(also known as “TROLOX”), porphyrin or metalloporphyrin antioxidants,particularly manganese porphyrin superoxide dismutase/catalase mimeticssuch as Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin(MnTE-2-PyP) (see, e.g., R. Rosenthal et al., J. Biol. Inorg. Chem. 14:979-991 (2009); see also U.S. Pat. No. 5,994,339 to Crapo et al.; U.S.Pat. No. 6,127,356 to Crapo et al.; and U.S. Pat. No. 8,067,405 toKlimko et al.), phenols, propyl gallate, flavonoids and/or naturallyoccurring substrates containing flavonoids, hydroxylated derivatives ofthe flavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen, etc., including combinationsthereof, See, e.g., U.S. Pat. Nos. 7,999,003 and 5,928,654.

Microcapsules of the present invention may be administered afterproduction, refrigerated and/or cryopreserved for subsequent use, and/orcultured for subsequent use, as desired. Microcapsules of the inventionmay be washed (e.g., in sterile physiological saline solution) prior toformulation and/or administration, as needed depending upon their mannerof production.

4. Formulation, Administration and Uses.

Microcapsules of the present invention may be administered per se orformulated for administration by any suitable technique, such as bymixing with sterile physiological saline solution. The microcapsules maybe administered by any suitable technique, including but not limited tosurgical implantation or injection (either of which may be carried outsubcutaneously, intraperitoneally, intramuscularly, or into any othersuitable compartment. Dosage of cells administered can be determined inaccordance with known techniques or variations thereof that will beapparent to those skilled in the art. For comparison, in the treatmentof diabetes, the International Islet Transplant Registry has recommendedtransplants of at least 6,000 cells per kilogram of recipient bodyweight, to achieve euglycemia. In the present invention, the number ofcells implanted will depend upon the age and condition of the subject,the particular disorder being treated, etc. In some embodiments of thepresent invention, from 1,000, 2,000, 3,000, or 6,000 cells per kilogramof recipient body weight, up to 20,000, 40,000 or 60,000 cells perkilogram recipient body weight, are administered.

While the present invention is described primarily with reference to theencapsulation of pancreatic islet cells for the treatment of diabetes(including type I and type II diabetes), it will be appreciated that avariety of different cell types can advantageously be co-encapsulatedwith oxygen-generating particles in the manner described herein. Forexample, any of a variety cells types, particularly stem cells (e.g.,mesenchymal stem cells isolated from bone marrow, muscle tissues,dermis, or combinations thereof) for encapsulation in injectableparticles for tissue construction, reconstruction or repair, asdescribed in U.S. Pat. No. 7,338,657 to Vogel.

Microcapsules of the present invention that contain ovarian granulosaand theca cells may be administered to subjects as a treatment for anycondition in which estrogen replacement therapy is used. Subjects orpatients to be treated by such methods include subjects afflicted with,or at increased risk of, one or more of osteoporosis, hot flashes,irregular period, vaginal atrophy, vaginal and/or bladder infection,incontinence (e.g., urge incontinence, stress incontinence), fatigue,sleep disturbances, irritability, mood swings, depression, loss ofmuscle mass, increased fat tissue, thinning and loss of skin elasticity,loss of bone tissue, impaired cognition etc., which may be associatedwith menopause, hysterectomy, ovarectomy, or other condition for whichestrogen or hormone replacement therapy is employed.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1 Co-Encapsulation of Islet Cells with Particulate OxygenGenerating Substances (POGS)

Transplanted islets are subjected to extended periods of hypoxiafollowing the process of isolation, purification, and transplantation,which affects their survival rates. The goal of the present study was toexplore the potential use of particulate oxygen generating substances(POGS) to enhance the viability of encapsulated islets fortransplantation.

Materials:

Calcium Peroxide, CaO₂ (CPO), Magnesium Peroxide, MgO₂, (MPO), Catalase(from bovine liver), Trolox (Sigma-Aldrich, USA), Calcium chloride,HEPES, Zinc Sulfate, Histopaque-1083, and HBSS were purchased fromSigma-Aldrich (USA). CMRL-1066, and all reagents for the human isolationunless otherwise noted were purchased from Cellgro (Manassas, Va.). ITSplus medium was purchased from BD (Franklin Lakes, N.J.). L-glutamateand penicillin/streptomycin were purchased from Gibco (USA). ClzymeCollagenase was purchased from VitaCyte (Indianapolis, Ind.). Lowviscosity (20-200 mPa s) ultra-pure sodium alginate with high mannuronicacid content (LVM) were purchased from Nova-Matrix (Oslo, Norway) whichwas reported by the manufacturer to have molecular weights 75-200 kDaand a G/M ratio of ≦1.

Human Islet Isolation, Purification and Culture:

Human islets were isolated from cadaveric human pancreases using theRicordi technique (J. Lakey et al., Current Human Islet IsolationProtocol. 1 edn, (Medical Review Co. 2004)) with collagenase digestionof tissue, and following purification using a COBE 2991 and a histopaquegradient, were maintained for 24 hours under standard cell cultureconditions in Memphis Serum Free Media (MSFM)(A. Gaber and D. Fraga,Advances in long-term islet culture—The Memphis experience. CellBiochem. Biophys., 49-54 (2004)) prior to encapsulation.

Comparison of CPO and MPO:

We first examined the oxygen release profiles of two POGS, CPO and MPOencapsulated in alginate microbeads in the absence or presence ofcatalase (100 U/ml), and the antioxidant, Trolox (1.5 mM). Sieves wereused to select both MPO and CPO particles equal to and less than 25 μmin diameter. Then 0.5% by weight solution of 1.5% LVM and either CPO orMPO were prepared respectively. These solutions were then extrudedthrough an encapsulating device forming microspheres measuring 300 to400 μM in diameter, which were allowed to crosslink for 15 minutes in1.1% CaCl₂ solution. After cross-linking, the microbeads were rinsedwith HBSS and placed into 50 ml conical tubes (BD, USA) containing 20 mLultra pure water with 10 mM HEPES (pH=7.4). Tubes were placed in a 1%oxygen environment in a hypoxic glovebox system (Biospherix, USA) withthe caps of the tubes removed allowing for gas exchange. Dissolvedoxygen readings were taken every 24 hours for 6 days using an Orion4-star DO Probe (Thermo Science).

Oxygen Release from MPO Alginate Capsules:

On the basis of a better release profile, we then selected MgO₂ forfurther studies. First the release profiles of various concentrations ofMPO were measured. Using the same technique as described above 0%,0.25%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 5%, 10%, 20%, 30%, and 40% (byweight) solutions of MPO in 1.5% UP LVM were prepared, with particleslarger than 25 μM sieved out of the solution prior to encapsulation. TheMPO particles in the solutions were then encapsulated and placed intothe hypoxic chamber in 50 ml conical tubes. Dissolved oxygen levels weremeasured for up to 250 hours. Using the same methodology we thenassessed the effect of the antioxidants, Catalase and Trolox, on oxygenrelease. A 2.5% solution of MPO and 1.5% LVM was prepared, and was mixedwith 50, 100, and 200 U/ml of catalase, 0.5, 1.0, and 1.5 mM Troloxrespectively. Capsules were prepared and dissolved oxygen levels weremeasured as described earlier. Lastly the combined effects of Trolox andCatalase were investigated; capsules were prepared with 2.5% MPO, and1.5% LVM and 0.5 mM+50 U/ml, 1.5 mM+100 U/ml, and 3.0 mM+200 U/ml Troloxand Catalase respectively. Dissolved oxygen levels were measured for upto 250 hours.

Islet Encapsulation:

After 24 hours of culture, islets were mixed at a concentration of 1000islets/ml with 1.5% ultra pure low-viscosity high mannuronic acid (LVM)alginate (with or without 2.5% MgO₂, 100 U/ml catalase and 1.5 mMTrolox). Each islet suspension was then separately extruded through amicrofluidic device at a flow rate of 1.4 ml/min and an air pressure of15 psi to generate microbeads with a mean±SD diameter of 400±100 μm. Themicrobeads were allowed to crosslink for 15 minutes in a solution of1.1% CaCl₂ and 10 mM HEPES and were then rinsed with HBSS and culturedin MSFM for 24 hours under standard culture conditions. Naked islets(control) and encapsulated islets were stained for Live/Dead viabilityassay with carboxy-fluorescein diacetate (CFDA) and propidium iodide(PI) after an additional 24 hours culture. Confocal microscopy was usedto image the stained islets and viability was quantified by counting thepercentage of live cells over the total number of cells with Image J.Results were statistically analyzed using a one-way ANOVA with Tukeypost test.

Results and Discussion:

We found that MPO provided a higher release of oxygen for up to fivedays compared to CPO, FIG. 1. We found that the duration of oxygenrelease from alginate microbeads containing MgO₂ was significantlyshortened by the presence of catalase alone or in combination withTrolox; however there was no significant change in oxygen release by MPOin the presence of Trolox alone (FIG. 2). The % mean±SD viability ofnaked unencapsulated islets was significantly lower (7.1±6) thanencapsulated islets alone (47.4±5.6) (p<0.01, n=3). The viability ofencapsulated islets with MgO₂, Catalase, and Trolox was significantlyhigher (76.2±10.9) than that measured in encapsulated islets withoutMgO₂ (p<0.05) (FIG. 3).

Conclusion:

In summary, we found that the 3D environment of microbeads enhanced theviability of islets in culture and that MgO₂ enhanced encapsulated isletcell viability further in the presence of antioxidants, as oxygenreleased from POGS was readily available to the islets.

EXAMPLE 2 Inclusion of a SOD-mimicking Catalytic Antioxidant withCo-Encapsulated Islet Cells and POGS

Previous studies have shown that the isolation process predisposesislets to subsequent damage and functional impairment and that an SODmimicking catalytic antioxidant, MnTE-2-PyP (also called AEOL10113),developed by Drs. Piganelli and Crapo, can be beneficial in preservingislet cell functional mass when used in the isolation or culture medium(R. Bottino et al., Preservation of human islet cell functional mass byantioxidative action of a novel SOD mimic compound. Diabetes 51: 2561(2002); R. Bottino et al., Diabetes 53: 2559 (2004)). We have found thatMnTE-2-PyP significantly (p<0.05) enhances the viability of isolatedhuman islets when added to islets in normal culture (FIG. 4). In FIG. 4,we still further show that MnTE-2-PyP counteracts the diminishing effectof MPO on islet viability.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A microcapsule comprising: (a) a liquidaqueous or hydrogel core; (b) a semipermeable membrane surrounding saidcore; (c) live mammalian pancreatic islet cells in said core; and (d)oxygen-generating particles in said core, said oxygen-generatingparticles included in said microcapsules in an amount sufficient tolengthen the duration of viability of said mammalian animal cells insaid microcapsules wherein said oxygen-generating particles areinorganic peroxides selected from the group consisting of calciumperoxide, magnesium peroxide, sodium peroxide, sodium percarbonate,benzylperoxide, and combinations thereof.
 2. The microcapsule of claim1, wherein said oxygen-generating particles further comprise a radicaltrap or peroxide decomposition catalyst.
 3. The microcapsule of claim 1,wherein said microcapsule further comprises an antioxidant.
 4. Themicrocapsule of claim 3, wherein said antioxidant is a metalloporphyrinantioxidant.
 5. The microcapsule of claim 1, said microcapsule furthercomprising an auxiliary layer surrounding said semipermeable layer. 6.The microcapsule of claim 5, wherein said auxiliary layer comprises abiodegradable polymer, said biodegradable polymer optionally containingan active compound.
 7. The microcapsule of claim 6, wherein saidauxiliary layer contains a pro-angiogenic agent in an amount effectiveto enhance the growth of blood vessels adjacent said microcapsule.
 8. Acomposition comprising a plurality of microcapsules of claim
 1. 9. Thecomposition of claim 8, further comprising a pharmaceutically acceptablecarrier.
 10. A method of treating diabetes in a subject in need thereof,comprising implanting microcapsules of claim 1 in said subject in atreatment-effective amount.
 11. A microcapsule comprising: (a) a liquidaqueous or hydrogel core; (b) a semipermeable membrane layer surroundingsaid core; (c) live mammalian pancreatic islet cells in said core; (d)oxygen-generating particles in said core, said oxygen-generatingparticles included in said microcapsules in an amount sufficient tolengthen the duration of viability of said mammalian cells in saidmicrocapsules; wherein said oxygen-generating particles comprise (i) aninorganic peroxide, optionally in combination with a biodegradablepolymer comprising alginate, and (ii) a radical trap or peroxidedecomposition catalyst; (e) an antioxidant; and (f) an auxiliary layersurrounding said semipermeable membrane layer, wherein said auxiliarylayer comprises a biodegradable polymer, said biodegradable polymercontaining an angiogenic agent in an amount effective to enhance thegrowth of blood vessels adjacent said microcapsule wherein saidoxygen-generating particles are inorganic peroxides selected from thegroup consisting of calcium peroxide, magnesium peroxide, sodiumperoxide, sodium percarbonate, benzylperoxide, and combinations thereof.12. The microcapsule of claim 11, wherein said antioxidant is ametalloporphyrin antioxidant.
 13. The microcapsule of claim 11, wherein:said organic or inorganic peroxide is magnesium peroxide; saidbiodegradable polymer of said oxygen generating particles is present andis comprised of alginate; said radical trap or peroxide decompositioncatalyst is catalase; said antioxidant is trolox; said auxiliary layeris comprised of sodium alginate; and said angiogenic agent is vascularendothelial growth factor.